Role of the epstein-barr virus RTA protein in activation of distinct classes of viral lytic cycle genes

Abstract

Initiation of the Epstein-Barr virus (EBV) lytic cycle is controlled by two immediate-early genes, BZLF1 and BRLF1. In certain epithelial and B-cell lines, their protein products, ZEBRA and Rta, stimulate their own expression, reciprocally stimulate each other's expression, and activate downstream viral targets. It has been difficult to examine the individual roles of these two transactivators in EBV-infected lymphocytes, as they are expressed simultaneously upon induction of the lytic cycle. Here we show that the Burkitt lymphoma cell line Raji represents an experimental system that allows the study of Rta's role in the lytic cycle of EBV in the absence and presence of ZEBRA. When expressed in Raji cells, exogenous Rta does not activate endogenous BZLF1 expression, yet Rta remains competent to transactivate certain downstream viral targets. Some genes, such as BaRF1, BMLF1, and a late gene, BLRF2, are maximally activated by Rta itself in the absence of detectable ZEBRA. The use of the Z(S186A) mutant form of ZEBRA, whose transactivation function is manifest only by coexpression of Rta, allows identification of a second class of lytic cycle genes, such as BMRF1 and BHRF1, that are activated in synergy by Rta and ZEBRA. It has already been documented that of the two activators, only ZEBRA stimulates the BRLF1 gene in Raji cells. Thus, there is a third class of viral genes activated by ZEBRA but not Rta. Moreover, ZEBRA exhibits an inhibitory effect on Rta's capacity to stimulate the late gene, BLRF2. Consequently ZEBRA may function to repress Rta's potential to activate some late genes. Raji cells thus allow delineation of the combinatorial roles of Rta and ZEBRA in control of several distinct classes of lytic cycle genes.

FIG. 1

Rta and ZEBRA stimulate the BRLF1 and BZLF1 genes to different extents in HH514-16 and Raji cells. Northern blot analysis of total cellular RNA isolated 30 h after chemical treatment or transfection is shown. Cells were either untreated (lanes 1 and 7), induced with TPA and sodium butyrate (lanes 2 and 8), or transfected with empty vector (lanes 3, 5, 9, and 11) or Rta and ZEBRA expression vectors (lanes 4, 6, 10, and 12). Northern blots were probed with a 30-base oligonucleotide from within exon I of BZLF1, which detects the 1.0-kb mRNA originating at Zp, the 3.0-kb bicistronic message from Rp, and a 1.3-kb vector transcript from BXG1/genZ. A probe for the H1 component of RNase P was used to control for RNA loading. (A) HH514-16 cells; (B) Raji cells.

FIG. 2

Immunoblots comparing activation of lytic cycle genes following the transfection of Rta and ZEBRA into Raji cells. Cells were either untreated (lane 1), chemically induced with TPA and sodium butyrate (T/B; lane 2), or transfected with 10 μg of plasmid DNA (lanes 3 to 7). In lanes 4 and 6, cells received 5 μg of activator (ZEBRA [Z] or Rta [R]) and 5 μg of empty vector. In lanes 3 and 5, cells received only vector pBXG1 (V1; lane 3) or pRTS (V2; lane 5). In lane 7, vectors expressing both ZEBRA and Rta were transfected. Immunoblots prepared 72 h following transfection were probed sequentially with the indicated antisera for EBNA1, Rta, EA-D, ZEBRA, BLRF2 (LR2), and BFRF3 (FR3).

FIG. 3

Rta does not activate ZEBRA expression in Raji cells. (A) Reporter assay. Raji cells were transfected with 16 μg of total plasmid DNA consisting of the indicated amount of Rta or ZEBRA expression vector (made up to 5 μg with pBXG1; U, only pBXG1), 10 μg of E4CAT or Z3E4CAT reporter vector, and 1 μg of luciferase control vector. CAT and luciferase assays were performed 72 h following transfection. CAT activity was standardized for transfection efficiency on the basis of the luciferase data. Fold activation is the ratio of CAT activity generated by the reporter E4CAT or Z3E4CAT in the presence of Rta or ZEBRA divided by the activity in the presence of empty expression vector. (B) Western blot. Protein extracts made from the same experiment were probed on an immunoblot for EA-D and ZEBRA.

FIG. 4

EBV lytic cycle genes activated by Rta alone or together with Z(S186A). Cells were either untreated (lane 1), chemically induced with TPA and sodium butyrate (lane 2), or transfected with 10 μg of plasmid DNA (lanes 3 to 10). In lanes 4, 6, and 8, cells received 5 μg of activator and 5 μg of empty vector. In lanes 3, 5, and 7, cells received only vector pRTS (lane 3), pBXG1 (lane 5), or pCMV (lane 7). In lanes 9 and 10, Rta was transfected with ZEBRA and the mutant Z(S186A), respectively. Total RNA prepared 30 h following transfection was analyzed by Northern blotting using probes for the indicated genes (see Materials and Methods). The blot was stripped between probes. Classification of the genes according to primary activator(s) is indicated to the right (see Discussion). The extra band above the expected size of the BRLF1 mRNA in lane 10 is most likely the result of an Rta-activated transcript from the Z(S186A) expression vector.

FIG. 5

Comparison of Rp (A) and Zp (B) promoter sequences among Raji, HH514-16, and B95-8 genomes. Promoters were amplified from total Raji and HH514-16 cell DNA by PCR, and sequences were compared to that of the B95-8 prototype. Deviations from B95-8 and their locations relative to the transcriptional start site are indicated in bold; known regulatory sites affected by the mutations are indicated on the right. The diagrams represent the promoters with their known proximal regulatory elements. ZRE, ZIIIA, and ZIIIB, ZEBRA response elements; Z1 and ZIA through -D, AT-rich sequences, reported to bind Sp1, Sp3, and MEF2D (7, 33, 34); SRE, serum response element; OCT, AP-1-like octamer; TRE, TPA response element.

FIG. 6

Activation of RpCAT containing sequences from B95-8, HH514-16, and Raji genomes by Rta in Raji cells. See the legend to Fig. 3 for experimental details. Fold activation is the ratio of CAT activity generated by a reporter in the presence of Rta divided by the CAT activity in the presence of empty vector. pCAT, reporter lacking promoter or enhancer sequences; RpCAT, Rp from the three respective genomes cloned into pCAT.

FIG. 7

Rta activates BLRF2, a late gene. (A and B) HH514-16 cells; (C and D) Raji cells. (A and C) Western analysis of protein extracts from cells that were uninduced (lanes 1 and 2) or had been chemically induced (lanes 3 and 4) or transfected with 5 μg of empty vector (lanes 5, 6, 9, and 10) or 5 μg of expression vector (lanes 7, 8, 11, and 12). For each condition, the cells had also been either untreated (−) or treated with the viral DNA polymerase inhibitor PAA (+). Immunoblots were prepared 30 h following transfection and probed sequentially with antisera to the indicated proteins (LR2, BLRF2; FR3, BFRF3). (B and D) Northern analysis of RNA prepared from cells of the same transfections as above. The blots were probed with a ³²P-labeled oligonucleotide from within BLRF2 and a fragment of the H1 component of RNase P as loading control.